A growing body of evidence indicates that epigenetic mechanisms, including post-translational modifications (PTMs) of histone proteins, are associated with a number of psychiatric disorders including addiction. This project utilizes a mouse line expressing a tetO-regulated, HA-tagged histone H3.3 protein exclusively in forebrain neurons. The H3.3 variant is unique in that H3.3 incorporates into chromatin independently of DNA replication and preferentially at active sites of transcription and transcription factor binding. Thus, modifications of H3.3 and histones associated with H3.3-containing nucleosomes are particularly likely to be involved in plasticity such as following repeated exposure to cocaine. This proposal is a collaborative effort between the Pierce lab, which focuses on animal models of addiction, and the Garcia lab, which specializes in cutting-edge mass spectrometry (MS)-based quantification of histone modifications. Both specific aims utilize novel MS- based proteomics platforms to comprehensively interrogate the PTM profiles of histones during and following priming- (Aim 1) or cue- (Aim 2) induced reinstatement of cocaine seeking. All experiments will be performed in both male and female mice. Although certain histone modifications have been implicated in models of addiction, changes in combinatorial histone modifications in response to cocaine self-administration remain poorly understood. Quantitative MS offers the ability to study histone modifications in an unbiased fashion. Specific Aim 1 focuses on the cocaine self-administration/extinction/reinstatement model of relapse. Mice will be allowed to self-administer cocaine or will receive passive, yoked infusions of saline or cocaine. Following the extinction of cocaine seeking, mice will be challenged with a systemic injection of cocaine that promotes cocaine reinstatement or will be injected with saline. One hour following the cocaine or saline injection, the mice will be killed and the nucleus accumbens, a brain region associated with cocaine-induced neuronal plasticity, will be dissected and prepared for MS to quantify acutely altered histone PTMs. Specific Aim 2 focuses on combinatorial histone modifications associated with the incubation of cue-induced cocaine reinstatement. Following cocaine self-administration, the mice will undergo forced abstinence for 1 or 30 days. Some animals will be assessed for cue-induced reinstatement and then killed after a 60-minute session; control accumbens tissue will be collected in the absence of the reinstatement test. Alterations in histone PTMs subsequently will be assessed via MS as in Aim 1. We hypothesize that histone acetylation and phosphorylation will increase in active regions of the neuronal genome particularly during and immediately following cocaine reinstatement (priming or cue-induced reinstatement, as assessed in Aims 1 and 2, respectively). In contrast, we expect to observe increases in histone methylation associated with reduced gene expression and decreases in histone methylation in regions of active transcription that persist through the extended abstinence period following cocaine self-administration in Aim 2.